PIGF is involved in important physiological and pathological processes, in particular angiogenesis. It plays an important part in tumor progression, kidney diseases, which are caused in particular by diabetes mellitus, in psoriasis, inflammatory diseases, in particular rheumatoid arthritis, in cardiovascular diseases and the like [Iyer, S.; Leonidas, D. D.; Swaminathan, G. J.; Maglione, D.; Battisti, M.; Tucci, M.; Persico, M. G.; Acharya, K. R. J Biol Chem 2001, 276, (15), 12153-61./Iyer, S.; Acharya, K. R. Trends Cardiovasc Med 2002, 12, (3), 128-34./Heeschen, C.; Dimmeler, S.; Fichtlscherer, S.; Hamm, C. W.; Berger, J.; Simoons, M. L.; Zeiher, A. M. JAMA 2004, 291, (4), 435-41./Yang, W.; Ahn, H.; Hinrichs, M.; Torry, R. J.; Torry, D. S. J Reprod Immunol 2003, 60, (1), 53-60.].
PIGF is mainly expressed in the placenta and belongs to the “cysteine-knot” protein family. PIGF occurs in different forms. Different forms of PIGF are (I) primary isoforms and (II) secondary isoforms. (III) In addition, a distinction can be made between free PIGF (fPIGF) and bound PIGF (gPIGF).
(I) Primary PIGF Isoforms
Primary PIGF isoforms are characterized by the primary sequence, i.e. the order of the amino acids in the protein. Alternative splicing and posttranslational modifications, such as glycosylations, phosphorylations, degradation (degradation products, fragments, etc.), acetylations etc., lead to different primary PIGF isoforms. To date, four different primary isoforms of human PIGF, PIGF-1 (PIGF-131), PIGF-2 (PIGF152), PIGF-3 (PIGF-203) and PIGF-4 have been described.
The sequence of the PIGF-1 precursor (Sequence number (SN) 1V) is as follows:
SN 1V(SEQ ID NO: 1)1 61VVSEYPSEVE HMFSPSCVSL LRCTGCCGDE NLHCVPVETA NVTMQLLKIR SGDRPSYVEL 121
Secreted PIGF-1 does not as a rule possess the leader sequence of the PIGF-1 precursor (PIGF precursor) and thus begins at the N-terminus with alanine (A) (stated as  in the sequence of the PIGF-1 precursor, see above). This as a rule applies also to the other primary PIGF isoforms.
The sequence of the primary PIGF-1 isoform is thus as follows:
SN 1:(SEQ ID NO: 2)1 61 121KPERCGDAVP RR
In this primary sequence, possible sites for post-translational modifications, and hence also the presence of posttranslationally modified primary isoforms, can be discerned. For example, in general in vivo the posttranslationally modified primary PIGF isoform of PIGF-1 glycosylated at position 84 (asparagine, ) is present.
As the first N-terminal amino acid of the primary isoform PIGF-1, methionine (M) is often stated instead of alanine. This in general relates to recombinantly, for example in Escherichia coli (E. coli), expressed PIGF-1(rPIGF-1), and in particular to the human rPIGF-1 (rhPIGF-1). Here AUG, which codes for methionine, is used as the start codon. Such a PIGF expressed in E. coli has no posttranslational modifications, in particular also no glycosylations.
The sequence of the recombinant, human, primary PIGF-1 isoform is generally stated as follows:
SN 1RH:(SEQ ID NO: 3)1 61VSLLRCTGCC GDENLHCVPV ETANVTMQLL KIRSGDRPSY VELTFSQHVR CECRPLREKM 121KPERCGDAVP RR
Through alternative splicing, the sequence RRRPKGRGKRRREKQRPTDCHL (SEQ ID NO: 64) is present in the PIGF-2 isoform instead of the arginine (R) 124. The sequence of the primary PIGF-2 isoform thus reads:
SN 2:(SEQ ID NO: 4)1ALPAVPPQQW ALSAGNGSSE VEVVPFQEVW GRSYCRALERLVDVVSEYPS EVEHMFSPSC 61VSLLRCTGCC GDENLHCVPV ETANVTMQLL KIRSGDRPSYVELTFSQHVR CECRPLREKM 121KPERRRPKGR GKRRREKQRP TDCHLCGDAV PRR
An insert of 72 amino acids inserted by alternative splicing (HSPGRQSPDMPGDFRADAPSFLPPRRSLPMLFRMEWGCALTGSQS AVWPSSPVPEEIPRMHPGRNGKKQQRK (SEQ ID NO: 65)) leads to the sequence of the primary PIGF-3 isoform:
SN 3:(SEQ ID NO: 5)1ALPAVPPQQW ALSAGNGSSE VEVVPFQEVW GRSYCRALER LVDVVSEYPS EVEHMFSPSC 61 121 181
The primary PIGF-4 isoform contains sequences both of the PIGF-2 isoform (italic) and also of the PIGF-3 isoform (underlined):
SN 4:(SEQ ID NO: 6)1ALPAVPPQQW ALSAGNGSSE VEVVPFQEVW GRSYCRALER LVDVVSEYPS EVEHMFSPSC 61 121 181(II) Secondary PIGF Isoforms
Secondary PIGF isoforms result from the combination of primary PIGF isoforms or other molecules, in particular molecules which are homologous to PIGF. The primary PIGF isoforms or other molecules are subunits of the secondary PIGF isoforms. In general, secondary PIGF isoforms consist of two subunits. Thus PIGF is as a rule present as a dimer, i.e. as a homodimer or a heterodimer. Homodimers consist of two identical primary PIGF isoforms (subunits) such as PIGF-1×PIGF-1, PIGF-2×PIGF-2, PIGF-3×PIGF-3 and PIGF-4×PIGF-4. Heterodimers consist of two different primary PIGF isoforms or of one primary PIGF isoform and one other molecule, in particular a PIGF homolog such as vascular endothelial growth factor (VEGF) and primary isoforms thereof. Possible examples of heterodimers are PIGF-1×PIGF-2, PIGF-3×PIGF-4, PIGF-1×VEGF, etc.
(III) Free PIGF (fPIGF) and Bound PIGF (gPIGF)
Since PIGF forms complexes with binding partners, the complexed or bound forms of PIGF must also be considered as well as the isoforms. In principle, the free primary, but in particular the free secondary PIGF isoforms (free PIGF, fPIGF), should be distinguished from the complexed or bound forms (bound PIGF, gPIGF). gPIGF is for example homodimeric PIGF-1 which is present in complexed form. These can be simple complexes, i.e. a PIGF-1 homodimer is bound to a receptor, for example the membrane-bound fms-like tyrosine kinase receptor-1 (mFlt-1). Other examples are complexes with the soluble Flt-1 (sFlt-1), with neurophilins (NP; in particular NP-1 and NP-2), with the kinase domain-containing receptor/fetal liver kinase receptor (KDR/Flk-1, VEGFR-2), with heparin sulfate proteoglycans (HSPG) and isoforms, homologs, fragments and degradation products thereof. Multilayer constituted complexes of several and sometimes different PIGF isoforms and several and sometimes different binding partners, in particular receptors, are also possible.
The function of PIGF is mediated, modulated or inhibited by binding to the membrane-bound or soluble fms-like tyrosine kinase receptor-1 (fms-like tyrosine kinase receptor-1 (Flt-1) or Vascular Endothelial Growth Factor (VEGF) receptor-1 (VEGFR-1)) and the kinase domain-containing receptor/fetal liver kinase receptor (KDR/Flk-1 or VEGFR-2). In addition to other possible functions of PIGF, the binding of PIGF to membrane-bound Flt-1 (mFlt-1) is especially important. This results in mFlt-1 transphosphorylation and thus activates signal transduction cascades [Iyer, S.; Acharya, K. R. Trends Cardiovasc Med 2002, 12, (3), 128-34.].
In contrast to this, it is presumed that the binding of PIGF to soluble Flt-1 (sFlt-1) serves to reduce the physiological activity of PIGF [Iyer, S.; Acharya, K. R. Trends Cardiovasc Med 2002, 12, (3), 128-34.]. Furthermore it is presumed that the PIGF isoform is involved. PIGF-2, which is possibly linked to the membrane, has a cationic insert of 21 amino acids at the carboxy terminal end. Through the binding of anionic, in particular polyanionic, substances such as heparin, heparin sulfate proteoglycans, etc., further functions can be mediated. The N-glycosylation of asparagine (Asn) 84 and the amino acid sequence which is present in PIGF-3 can also have similar effects. Moreover, it is presumed that the binding of PIGF and VEGF has yet another effect, since here the VEGF expression and thus its activity is negatively regulated [Iyer, S.; Acharya, K. R. Trends Cardiovasc Med 2002, 12, (3), 128-34.]. In summary, this means that the various forms of PIGF have different functions or exert different effects.
For the current detection methods and binding partners, in particular antibodies, which are at present used for analytical and diagnostic purposes, there is the problem that the different forms of PIGF are not, or not efficiently enough (not sufficiently specifically) distinguished. For example, the “Anti-human PIGF Antibody” from R&D Systems, Inc. does not exclusively recognize certain PIGF forms, in particular rhPIGF-1 homodimer but also the heterodimer of rhPIGF and VEGF and rhPIGF-2 (R&D Systems Catalog Number: AF-264-PB or DPG00 product descriptions).
Furthermore, fPIGF or gPIGF are not exclusively detected, i.e. with the existing antibodies, a distinction between fPIGF and gPIGF is not or not sufficiently efficiently made. In particular, the specific detection of fPIGF is inadequate. This is demonstrated by the fact that rhFlt-1 in the form of rhFlt-1/Fc has an effect on the determination of PIGF (R&D Systems Catalog Number: DPG00). This non-specificity is confirmed in the literature [Maynard, S. E.; Min, J. Y.; Merchan, J.; Lim, K. H.; Li, J.; Mondal, S.; Libermann, T. A.; Morgan, J. P.; Sellke, F. W.; Stillman, I. E.; Epstein, F. H.; Sukhatme, V. P.; Karumanchi, S. A. J Clin Invest 2003, 111, (5), 649-58.]. Maynard et al. show that the relevant R&D Systems ELISA (R&D Systems Catalog Number: AF-264-PB or DPG00) does show a certain specificity for fPIGF, however the studies performed show that this specificity is low. In the determination of 0.5 ng/mL rhPIGF-1, a signal reduction of only about 12% is to be seen in the presence of 0.5 ng/mL sFlt-1. Even with a 10-fold excess of sFlt-1 (5 ng/mL), there was only a signal reduction by the factor of 2. A more pronounced signal reduction would occur with higher specificity of the antibodies used towards fPIGF.